Genomic and Transcriptomic Landscape of RET Wild-Type Medullary Thyroid Cancer and Potential Use of Mitogen-Activated Protein Kinase-Targeted Therapy.

BACKGROUND: About 75% of medullary thyroid cancers (MTCs) are sporadic with 45-70% being driven by a RET mutation. Selpercatinib is an approved treatment for RET-mutated (mutRET) MTC, however, treatments are needed for wild-type RET MTC (wtRET). Genomic alterations and transcriptomic signatures of wtRET MTC may reveal new therapeutic insights. METHODS: We did a retrospective analysis of MTC samples submitted for DNA/RNA sequencing and PD-L1 expression using IHC at a CLIA/CAP-certified lab. Tumor microenvironment immune cell fractions were estimated using RNA deconvolution (quanTIseq). Transcriptomic signatures of inflammation and MAP kinase pathway activation scores (MPAS) were calculated. Mann-Whitney U, chi-square, and Fisher exact tests were applied (p-values adjusted for multiple comparisons). RESULTS: The 160-patient cohort included 108 mutRET and 52 wtRET MTC samples. wtRET tumors frequently harbored MAPK pathway mutations, including HRAS (42.31%), KRAS (15.7%), NF1 (6.7%), and BRAF (2%) whereas only one MAPK pathway mutation (NF1) was identified among mutRET MTC. Recurrent mutations seen in wtRET MTC included MGA, VHL, APC, STK11, and NFE2L2. Increased transcriptional activation of the MAPK pathway was observed in wtRET patients harboring mutations in MAPK genes. While the frequency of PD-L1 expression was similar in wtRET and mutRET (10.2% vs 7.0%, p=0.531), wtRET tumors were more often TMB-high (7.7% vs 0.0%, p=0.011), and wtRET MTC exhibited higher expression of immune checkpoint genes. CONCLUSIONS: We identified molecular alterations and immune-related features that distinguish wtRET from mutRET MTC. While RET mutation drives MTC in the absence of other alterations, we showed that wtRET MTC frequently harbors MAPK pathway mutations. These findings may indicate a potential basis for MAPK-targeted therapy, possibly in combination with oncology immune-oncology agents for selected patients with wtRET MTC.

Molecular profiling of gestational trophoblastic neoplasia: Identifying therapeutic targets.

OBJECTIVE: The treatment for high risk or recurrent gestational trophoblastic neoplasia (GTN) is a highly toxic multi-agent chemotherapy. For patients with progressive or recurrent GTN, checkpoint inhibitors have demonstrated anti-tumor activity; however, identification of novel therapies for GTN remain an unmet need. Therefore, we sought to characterize the molecular landscape of GTN to identify potential therapeutic targets. METHODS: GTN samples were analyzed using a combination of molecular - next-generation sequencing (NGS) or whole exome sequencing (WES)- and protein- Immunohistochemistry (IHC) analyses. GTN samples encompassed complete moles, choriocarcinoma, epithelioid trophoblastic tumors (ETT), and placental site trophoblastic tumors (PSTT). RESULTS: We analyzed 30 cases of GTN including 15 choriocarcinoma, 7 ETT, 5 PSTT, 1 invasive mole and 2 mixed histologies. The median age was 41.5. GTN samples were found to be PD-L1 positive (92.3%), tumor mutational burden (TMB) low (92.8%), and microsatellite stable (MSS) (100%). Forty-six percent of choriocarcinoma specimens contained a genomic alteration including TP53 (33%) and homologous recombination repair (HRR) (13%) genes. Alterations in RTK-RAS pathway signaling was present in 40% of ETT cases. CONCLUSIONS: The high rate of PD-L1 positivity in this real-world database and reported in prior literature support continued clinical trial development evaluating immunotherapy for treatment of GTN. Other potential targeted treatments identified include Wee1, PARP and MEK inhibitors based on molecular alterations in TP53, HRR genes, and RTK-RAS pathways respectively.

The Genomic Landscape of Vulvar Squamous Cell Carcinoma.

Vulvar squamous cell cancer (VSC) accounts for 90% of vulvar cancers. Next-generation sequencing studies of VSC imply human papillomavirus (HPV) and p53 status play separate roles in carcinogenesis and prognosis. We sought to describe the genomic landscape and analyze the immunologic profiles of VSC with respect to HPV and p53 status. A total of 443 VSC tumors underwent tumor profiling. Next-generation sequencing was performed on genomic DNA isolated from formalin-fixed paraffin-embedded tumor samples. PD-L1, microsatellite instability were tested by fragment analysis, IHC, and next-generation sequencing. Tumor mutational burden-high was defined as >10 mutations per MB. HPV 16/18 positive (HPV+) status was determined using whole exome sequencing on 105 samples. Three cohorts were identified from 105 samples with known HPV: HPV+, HPV-/p53wt, and HPV-/p53mt. Where HPV and p53 status were examined, TP53 mutations were exclusive of HPV+ tumors. In all, 37% of samples were HPV+. Among the 66 HPV- tumors, 52 (78.8%) were HPV-/p53mt and 14 (21.2%) were HPV-/p53wt. The HPV-/p53wt cohort had a higher rate of mutations in the PI3KCA gene (42.9% HPV-/p53wt vs 26.3% HPV+ vs. 5.8% HPV-/p53mt, q =0.028) and alterations in the PI3K/AkT/mTOR pathway (57.1% HPV-/p53wt vs. 34.2% HPV+ vs. 7.7% HPV-/p53mt, q =0.0386) than the other 2 cohorts. Ninety-eight VSC tumors with HPV16/18 information underwent transcriptomic analysis and immune deconvolution method. No differences were observed in immune profiles. The HPV-/p53wt VSC tumors had significantly higher rates of mutations in the PI3KCA gene and alterations in the PI3K/AkT/mTOR pathway, a potential target that merits further investigation in this subgroup.

Targeting de novo lipogenesis and the Lands cycle induces ferroptosis in KRAS-mutant lung cancer.

Mutant KRAS (KM), the most common oncogene in lung cancer (LC), regulates fatty acid (FA) metabolism. However, the role of FA in LC tumorigenesis is still not sufficiently characterized. Here, we show that KMLC has a specific lipid profile, with high triacylglycerides and phosphatidylcholines (PC). We demonstrate that FASN, the rate-limiting enzyme in FA synthesis, while being dispensable in EGFR-mutant or wild-type KRAS LC, is required for the viability of KMLC cells. Integrating lipidomic, transcriptomic and functional analyses, we demonstrate that FASN provides saturated and monounsaturated FA to the Lands cycle, the process remodeling oxidized phospholipids, such as PC. Accordingly, blocking either FASN or the Lands cycle in KMLC, promotes ferroptosis, a reactive oxygen species (ROS)- and iron-dependent cell death, characterized by the intracellular accumulation of oxidation-prone PC. Our work indicates that KM dictates a dependency on newly synthesized FA to escape ferroptosis, establishing a targetable vulnerability in KMLC.

Transcriptional Profiling of Malignant Melanoma Reveals Novel and Potentially Targetable Gene Fusions.

Invasive melanoma is the deadliest type of skin cancer, with 101,110 expected cases to be diagnosed in 2021. Recurrent BRAF and NRAS mutations are well documented in melanoma. Biologic implications of gene fusions and the efficacy of therapeutically targeting them remains unknown. Retrospective review of patient samples that underwent next-generation sequencing of the exons of 592 cancer-relevant genes and whole transcriptome sequencing for the detection of gene fusion events and gene expression profiling. Expression of PDL1 and ERK1/2 was assessed by immunohistochemistry (IHC). There were 33 (2.6%) cases with oncogenic fusions (14 novel), involving BRAF, RAF1, PRKCA, TERT, AXL, and FGFR3. MAPK pathway-associated genes were over-expressed in BRAF and RAF1 fusion-positive tumors in absence of other driver alterations. Increased expression in tumors with PRKCA and TERT fusions was concurrent with MAPK pathway alterations. For a subset of samples with available tissue, increased phosphorylation of ERK1/2 was observed in BRAF, RAF1, and PRKCA fusion-positive tumors. Oncogenic gene fusions are associated with transcriptional activation of the MAPK pathway, suggesting they could be therapeutic targets with available inhibitors. Additional analyses to fully characterize the oncogenic effects of these fusions may support biomarker driven clinical trials.

The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair.

P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

Integrating a 92-Gene Expression Analysis for the Management of Neuroendocrine Tumors of Unknown Primary

Background: Neuroendocrine tumors (NETs) are rare tumors that can originate from any part of the body. Often, imaging or exploratory surgery can assist in the identification of the tumor primary site, which is critical to the management of the disease. Neuroendocrine tumors (NETs) of unknown primary constitute approximately 10-15% of all NETs. Determining the original site of the tumor is critical to providing appropriate and effective treatment. Methods: We performed a retrospective review of neuroendocrine tumors at our institution between 2012 and 2016 using a 92-gene cancer ID analysis. Results: 56 patients with NETs of unknown primary were identified. Samples for 38 of the 56 underwent the 92-gene cancer ID analysis. The primary site of the tumor was identified with >95% certainty in 35 of the 38 patients. Conclusion: The 92-gene cancer ID analysis identified a primary site in 92% of our NETs study cohort that previously had been unknown. The results have direct implications on management of patients with regard to FDA-approved treatment options.

Elevated leptin disrupts epithelial polarity and promotes premalignant alterations in the mammary gland.

Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation.

Characterization of Squamous Cell Lung Cancers from Appalachian Kentucky.

BACKGROUND: Lung cancer is the leading cause of cancer mortality in the United States (U.S.). Squamous cell carcinoma (SQCC) represents 22.6% of all lung cancers nationally, and 26.4% in Appalachian Kentucky (AppKY), where death from lung cancer is exceptionally high. The Cancer Genome Atlas (TCGA) characterized genetic alterations in lung SQCC, but this cohort did not focus on AppKY residents. METHODS: Whole-exome sequencing was performed on tumor and normal DNA samples from 51 lung SQCC subjects from AppKY. Somatic genomic alterations were compared between the AppKY and TCGA SQCC cohorts. RESULTS: From this AppKY cohort, we identified an average of 237 nonsilent mutations per patient and, in comparison with TCGA, we found that PCMTD1 (18%) and IDH1 (12%) were more commonly altered in AppKY versus TCGA. Using IDH1 as a starting point, we identified a mutually exclusive mutational pattern (IDH1, KDM6A, KDM4E, JMJD1C) involving functionally related genes. We also found actionable mutations (10%) and/or intermediate or high-tumor mutation burden (65%), indicating potential therapeutic targets in 65% of subjects. CONCLUSIONS: This study has identified an increased percentage of IDH1 and PCMTD1 mutations in SQCC arising in the AppKY residents versus TCGA, with population-specific implications for the personalized treatment of this disease. IMPACT: Our study is the first report to characterize genomic alterations in lung SQCC from AppKY. These findings suggest population differences in the genetics of lung SQCC between AppKY and U.S. populations, highlighting the importance of the relevant population when developing personalized treatment approaches for this disease.

Immune checkpoint inhibitors in neuroendocrine tumors: A single institution experience with review of literature.

This unique case series and review of literature suggests that immune checkpoint inhibitors may have clinical activity in neuroendocrine tumors. OBJECTIVE: Summarize advances of immuno-oncology in neuroendocrine tumors with the help of a case series. DESIGN: Case series and review of literature. INTERVENTION OR EXPOSURE: The patients were treated with immune checkpoint inhibitors (pembrolizumab or nivolumab). MAIN OUTCOMES AND MEASURESS: Life expectancy, quality of life, disease progression. RESULTS: Maximum durable response of 16 months in one of the patients so far. All patients showed improvement in quality of life before disease progression. Two out of four are still on therapy. None of the patients experienced immune checkpoint inhibitor associated side-effects. All patients had failed standard of care therapy prior to the initiation of immune checkpoint inhibitors and were on the verge of hospice. CONCLUSIONS: Immune checkpoint inhibitors have revolutionized cancer management and the last 5 years have seen a rapid expansion in the indications for this class of drug. Neuroendocrine tumors, unfortunately, have been slow to catch on to the immuno-oncology, partly due to difficulties in establishing relevant preclinical neuroendocrine tumors models for immune-oncology studies. In this manuscript, we review the current status of immunotherapy in neuroendocrine tumors.

Prostate Cancer Biomarkers: Current Status.

Prostate cancer (PCa) is the second most frequently diagnosed cancer among men worldwide. Given the biological heterogeneity in localized PCa and its variable clinical course, a personalized approach to patient risk stratification and management is needed. A variety of high-throughput technologies, such as next-generation sequencing, transcriptomic, epigenetic, and metabolomic modalities have led to an improved understanding of the genomic basis of PCa and the identification of PCa biomarkers. Novel genomic approaches offer additional information to improve clinical decision making. The goal of this report is to review the use of currently available molecular biomarkers in the diagnosis and prognostication of PCa outcome.

The utility of tetraspanin CD9 as a biomarker for metastatic clear cell renal cell carcinoma.

The use of tetraspanin CD9 as a biomarker for renal cell carcinomas (RCC) has been explored with minor conclusions. Identification of a biomarker that not only distinguishes between the different types of renal cell carcinomas, but also predicts the metastatic potential of these tumors would significantly advance diagnosis and prognosis of kidney cancers. We utilized established cell lines to better understand the contribution of CD9 to the metastatic potential of clear cell renal cell carcinomas, and then applied our findings to the TCGA database and immunohistochemical analysis of human samples based on tumor grading to determine the utility of CD9 as a biomarker for RCC. Clear cell renal cell carcinoma (ccRCC) cell expression of tetraspanin CD9 was compared to normal kidney cells and found to be elevated. Upon knockdown of CD9, ccRCC cells obtained a more metastatic phenotype. We found E-cadherin expression to be repressed and the endothelial to mesenchymal transition markers Snail, Twist1, and Zeb1 to be elevated upon CD9 knockdown. Upon observing these gene expression changes in the TCGA database and in 10 cases, we found that CD9 and E-cadherin expression was lowered in higher grade ccRCC tumors. There was a significant correlation between CD9 and either E-cadherin, Snail, or Zeb1 in these tumors. Collectively, using tetraspanin CD9 in tandem with E-cadherin as a biomarker in renal cell carcinoma will help to not only distinguish between types, but also predict the metastatic potential of RCC.

Cotargeting Polo-Like Kinase 1 and the Wnt/ß-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer.

The Wnt/ß-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the ß-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/ß-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear ß-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/ß-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the ß-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of ß-catenin by enhancing binding between glycogen synthase kinase 3ß (GSK3ß) and ß-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.

Inhibition of polo-like kinase 1 (Plk1) enhances the antineoplastic activity of metformin in prostate cancer.

The widely used anti-diabetic drug metformin has been shown to exert strong antineoplastic actions in numerous tumor types, including prostate cancer (PCa). In this study, we show that BI2536, a specific Plk1 inhibitor, acted synergistically with metformin in inhibiting PCa cell proliferation. Furthermore, we also provide evidence that Plk1 inhibition makes PCa cells carrying WT p53 much more sensitive to low-dose metformin treatment. Mechanistically, we found that co-treatment with BI2536 and metformin induced p53-dependent apoptosis and further activated the p53/Redd-1 pathway. Moreover, we also show that BI2536 treatment inhibited metformin-induced glycolysis and glutamine anaplerosis, both of which are survival responses of cells against mitochondrial poisons. Finally, we confirmed the cell-based observations using both cultured cell-derived and patient-derived xenograft studies. Collectively, our findings support another promising therapeutic strategy by combining two well tolerated drugs against PCa proliferation and the progression of androgen-dependent PCa to the castration-resistant stage.

Atypical ductal hyperplasia: interobserver and intraobserver variability.

Interobserver reproducibility in the diagnosis of benign intraductal proliferative lesions has been poor. The aims of the study were to investigate the inter- and intraobserver variability and the impact of the addition of an immunostain for high- and low-molecular weight keratins on the variability. Nine pathologists reviewed 81 cases of breast proliferative lesions in three stages and assigned each of the lesions to one of the following three diagnoses: usual ductal hyperplasia, atypical ductal hyperplasia and ductal carcinoma in situ. Hematoxylin and eosin slides and corresponding slides stained with ADH-5 cocktail (cytokeratins (CK) 5, 14. 7, 18 and p63) by immunohistochemistry were evaluated. Concordance was evaluated at each stage of the study. The interobserver agreement among the nine pathologists for diagnosing the 81 proliferative breast lesions was fair (κ-value=0.34). The intraobserver κ-value ranged from 0.56 to 0.88 (moderate to strong). Complete agreement among nine pathologists was achieved in only nine (11%) cases, at least eight agreed in 20 (25%) cases and seven or more agreed in 38 (47%) cases. Following immunohistochemical stain, a significant improvement in the interobserver concordance (overall κ-value=0.50) was observed (P=0.015). There was a significant reduction in the total number of atypical ductal hyperplasia diagnosis made by nine pathologists after the use of ADH-5 immunostain. Atypical ductal hyperplasia still remains a diagnostic dilemma with wide variation in both inter- and intraobserver reproducibility among pathologists. The addition of an immunohistochemical stain led to a significant improvement in the concordance rate. More importantly, there was an 8% decrease in the number of lesions classified as atypical ductal hyperplasia in favor of usual hyperplasia; in clinical practice, this could lead to a decrease in the number of surgeries carried out for intraductal proliferative lesions.

Sarcomatoid carcinoma of the urinary bladder: the final common pathway of urothelial carcinoma dedifferentiation.

Sarcomatoid carcinoma of the urinary bladder is an unusual malignancy composed of both carcinomatous and sarcomatous components. It is an aggressive tumor that presents at an advanced stage and confers a much poorer prognosis than conventional urothelial carcinoma. The proper nomenclature and histogenesis of these tumors have been subjects of debate for some time. There is an emerging consensus that sarcomatoid carcinoma is the most appropriate term for these neoplasms. The recent World Health Organization classification has applied this term to all tumors showing morphologic and/or immunologic evidence of both malignant epithelial and mesenchymal differentiation. Such tumors have been postulated to represent either multiclonal collision tumors or monoclonal cancers with divergent differentiation; recent molecular studies favor the latter theory. In this study, we discuss the nomenclature, clinical features, pathology, differential diagnosis, molecular genetics, and histogenesis of sarcomatoid carcinoma. We emphasize the importance of molecular genetic studies in providing insight into the histogenesis of this neoplasm. Sarcomatoid carcinoma seems to represent the final common pathway of urothelial carcinoma dedifferentiation.

Urothelial lesions with inverted growth patterns: histogenesis, molecular genetic findings, differential diagnosis and clinical management.

A number of well-recognized urothelial lesions with inverted morphology occur in the urinary bladder. Some are so common that they are considered normal variants of urothelium, whereas others are rare. It is important for the surgical pathologist to recognize these lesions and their overlapping morphological features, because in some cases establishing an accurate diagnosis is challenging. In this article, we review the spectrum of inverted urothelial lesions of the bladder. Emphasis is placed on differential diagnosis, molecular genetic findings, morphology and histogenesis.